The roles of metal ions in proteins range from structural stabilization to catalysis. Design of metal-binding sites may therefore provide a route to novel proteins with interesting properties. Such sites could also be used to produce radiolabeled antibodies for diagnostic imaging of tumors and targeted radiotherapy as well as modulating the stability of therapeutic proteins. Metal-binding sites have been designed in the IgG-binding domain Bl of Streptococcal protein G. Optical absorption spectroscopy and circular dichroism suggest that Zn(ll) binds tetrahedrally with high affinity to three histidines and a cysteine as intended. The proposed research will focus on the characterization and modification of these novel sites. The specific aims of this proposal are: l. Detailed structural characterization of the designed variants by nuclear magnetic resonance. 2. Selection of variants more stable than the first generation designed variants by phage display technology. 3. Characterization of the stable variants by CD, optical absorption spectroscopy and NMR. Modification of the stable four- coordinate variants to three-coordinate variants, their characterization and assessment of their catalytic efficiency. These studies will enable us to understand the role of metal ions in protein structure, function and stability and provide fundamental principles for protein design.